Stress is believed to activate sphingomyelinase to generate ceramide, which serves as a second messenger in initiating the apoptotic response. Conclusive evidence for this paradigm, however, is lacking. In the present study, we used a genetic approach to address this issue directly. We show that lymphoblasts from Niemann-Pick patients, which have an inherited deficiency of acid sphingomyelinase activity, fail to respond to ionizing radiation with ceramide generation and apoptosis. These abnormalities are reversible up on restoration of acid sphingomyelinase activity by retroviral transfer of human acid sphingomyelinase cDNA. Acid sphingomyelinase knockout mice also expressed defects in radiation-induced ceramide generation and apoptosis in vivo. Comparison with p53 knockout mice revealed that acid sphingomyelinase-mediated apoptosis and p53-mediated apoptosis are likely distinct and independent. These genetic models provide definitive evidence for the involvement of acid sphingomyelinase in one form of stress-induced apoptosis.

Systemic or intratesticular release of TNF alpha and IL1 beta have been implicated in the reduced testosterone biosynthesis and impaired production of competent spermatozoa found in human patients suffering from sepsis or chronic inflammation. Although in vitro and in vivo studies have demonstrated that TNF alpha and IL1 beta intercept the hypothalamic-pituitary testis axis at different levels, the site(s) of action and relative contribution of each cytokine to the overall testicular failure associated to systemic inflammatory processes remains poorly defined. In this study we show that intratesticular delivery of TNF alpha induced a rapid (4 h) and sustained (up to 24 h) reduction in steroidogenic acute regulatory (StAR) protein expression and testosterone biosynthesis in nonstimulated or human chorionic gonadotropin-treated intact or hypophysectomized rats. Bilateral treatment with cell-permeant short-chain ceramides (C2-cer or C6-cer) reproduced the early (4 h) inhibitory action of TNFalpha on testosterone biosynthesis and testicular StAR expression. The inhibitory action of C2-cer or C6-cer was not observed in animals treated with inactive analogs (dihydroceramide), phosphorylcholine, sphingosine, or sphingosine-1P. In sharp contrast to the previously described ability of IL1 beta to prevent human chorionic gonadotropin-stimulated Leydig cell steroidogenesis in vitro, serum testosterone and testicular StAR protein expression remained unchanged in animals bilaterally injected with this cytokine. These data support the concept that TNF alpha triggers different effector mechanisms to directly inhibit Leydig cell StAR expression and steroidogenesis, which ultimately contribute to the global reproductive failure associated with chronic inflammation and sepsis.

In [3H]serine-labelled granulosa cells treatment with TNF alpha (10 ng/ml) resulted in a transient decrease in cellular [3H]sphingomyelin and generation of [3H]ceramide that remained elevated 60 min later. In cells labelled with [methyl-14C]choline, TNF alpha induced a similar reduction in [14C]sphingomyelin content that was accompanied by a sustained elevation in [14C]phosphorylcholine levels. In FSH-primed cells, TNF alpha inhibited P450-AROM activity in a dose-dependent manner, an effect that was also observed in cells treated with bacterial sphingomyelinase (SMase 0.003-0.3 U/ml) or increasing concentrations (0.1-10 microM) of N-acetylsphingosine (C2-cer) a membrane-permeable analogue of ceramide. These results support the notion that sphingomyelin degradation to a bioeffector molecule ceramide, may be an early event involved in TNF alpha-induced signal transduction in granulosa cells.

In granulosa cells labeled to isotopic steady-state with [3H]serine, addition of interleukin-1 beta (IL1 beta) or bacterial sphingomyelinase (SMase) induced a rapid decrease (approximately 60% by 10 min) in cellular [3H]Sphingomyelin content and a prolonged generation (up to 60 min) of [3H]ceramide, the immediate lipid-moiety generated in response to sphingomyelin hydrolysis. In FSH-treated cells, IL1 beta (0.3-30 ng/ml) inhibited progesterone biosynthesis in a dose-dependent manner, an effect that was also observed in cells exposed to increasing concentrations of bacterial SMase (0.003-0.3 U/ml) or the membrane-permeable ceramide analogue N-hexanoylsphingosine (C6-cer:0.1-10 microM). Abrogation of progesterone biosynthesis was not a sole consequence of inadequate cAMP biosynthesis because cyclic nucleotide levels remained elevated (3- to 4-fold over untreated cultures) after addition of IL1 beta, SMase, or two different cell permeable ceramide analogues (C2-cer and C6-cer) to gonadotropin-stimulated granulosa cells. Moreover, taken into account that exogenous SMase or C6-cer partially abolished progesterone biosynthesis induced by But2cAMP (0.5 mM) or cholera toxin (CTX: 1 microgram/ml), the above mentioned results support the notion that activation of the sphingomyelin pathway exerts its inhibitory effects on granulosa cell steroidogenic activity at site(s) of action both proximal and distal to cAMP generation. As determined by RT-PCR analysis, the inhibitory effect of IL1 beta, SMase, or C6-cer on gonadotropin-stimulated steroidogenesis was accompanied by arrested transcription of the mitochondrial cholesterol side chain cleavage enzyme (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5-4isomerase, the two FSH-inducible steps involved in progesterone biosynthesis. Although bacterial SMase or the ceramide analogue C6-cer alone did not exactly reproduce the effect of IL1 beta on granulosa cell prostaglandin E2 (PGE2) biosynthesis, both agents augmented net PGE2 production and messenger RNA levels of the inducible prostaglandin endoperoxide synthase/cyclooxygenase (PGHS-2) in cytokine-treated cells. Although the effect on PGHS-2 messenger RNA may account for the facilitatory role of ceramide on IL1 beta-induced PGE2 biosynthesis, neither SMase nor the membrane-permeant ceramide analogue were able to augment prostaglandin accumulation in the presence of exogenously added arachidonate precursor. Collectively, whereas these results show that ceramide triggers a negative-effector pathway that is both necessary and sufficient to reproduce the inhibitory effect of IL1 beta on FSH-stimulated granulosa cell steroidogenesis, they also support the notion that sphingomyelin hydrolysis may be important for cytokine-induced PGHS-2 expression but not sufficient to reproduce IL1 beta-stimulated PGE2 biosynthesis.

No abstract